Extracellular rage binding protein (EN-RAGE) and uses thereof

ABSTRACT

The present invention provides for an isolated human EN-RAGE peptide. The present invention also provides for a method for determining whether a compound is capable of inhibiting the interaction of an EN-RAGE peptide with a RAGE peptide, which comprises: (a) admixing: (i) a RAGE peptide or an sRAGE peptide or a fragment of either thereof, (ii) an EN-RAGE peptide or a fragment thereof, and (iii) the compound; (b) measuring the level of interaction between the peptide of step (a)(i) and the peptide of step (a)(ii), and (c) comparing the amount of interaction meausred in step (b) with the amount measured between the peptide of step (a) (i) and the peptide of step (a) (ii) in the absence of the compound, thereby determining whether the compound is capable of inhibiting the interaction of the EN-RAGE peptide with the RAGE peptide, wherein a reduction in the amount of interaction in the presence of the compound indicates that the compound is capable of inhibiting the interaction. The present invention also provides for a method for inhibiting inflammation in a subject which comprises administering to the subject a compound capable of interfering with the interaction between EN-RAGE peptide and receptor for advanced glycation endproduct (RAGE) in the subject thereby inhibiting inflammation in the subject.

The invention disclosed herein was made with Government support underNIH Grant No. AG00602 from the U.S. Department of Health and HumanServices. Accordingly, the U.S. Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byauthor and date within the text. Full citations for these publicationsmay be found listed alphabetically at the end of the specificationimmediately preceding the claims. The disclosures of these publicationsin their entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art asknown to those skilled therein as of the date of the invention describedand claimed herein.

The Receptor for AGE (RAGE) is a member of the immunoglobulinsuperfamily of cell-surface molecules (1–2). Originally identified andcharacterized as a cellular receptor for glucose (aldose sugar)-modifiedproteins, or Advanced Glycation Endproducts (AGEs) (3–13), RAGE hassubsequently been reported to interact with other ligands, in bothsettings of normal development and in Alzheimer's disease (14–16). Innormal development, RAGE interacts with amphoterin, a polypeptide whichmediates neurite outgrowth in cultured embryonic neurons. In thosestudies, either anti-RAGE F(ab′)₂ or soluble RAGE (sRAGE) inhibitedneurite outgrowth on amphoterin-coated matrices, but not on matricescoated with other substrates such as laminin or poly-l-lysine (3). Inlater studies, RAGE was identified as a receptor on neurons andmicroglia for amyloid-β-peptide, a polypeptide linked to thepathogenesis of neuronal toxicity and death in Alzheimer's disease.

SUMMARY OF THE INVENTION

The present invention provides for an isolated human EN-RAGE peptide.The present invention also provides for a method for determining whethera compound is capable of inhibiting the interaction of an EN-RAGEpeptide with a RAGE peptide, which comprises: (a) admixing: (i) a RAGEpeptide or an sRAGE peptide or a fragment of either thereof, (ii) anEN-RAGE peptide or a fragment thereof, and (iii) the compound; (b)measuring the level of interaction between the peptide of step (a) (i)and the peptide of step (a) (ii), and (c) comparing the amount ofinteraction meausred in step (b) with the amount measured between thepetpide of step (a) (i) and the peptide of step (a) (ii) in the absenceof the compound, thereby determining whether the compound is capable ofinhibiting the interaction of the EN-RAGE peptide with the RAGE peptide,wherein a reduction in the amount of interaction in the presence of thecompound indicates that the compound is capable of inhibiting theinteraction. The present invention also provides for a method forinhibiting inflammation in a subject which comprises administering tothe subject a compound capable of interfering with the interactionbetween EN-RAGE peptide and receptor for advanced glycation endproduct(RAGE) in the subject thereby inhibiting inflammation in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Immunohistochemistry of human kidney (active lupus nephritis).Kidney tissue from a patient with active lupus nephritis was obtained,fixed in formalin and paraffin-embedded sections were prepared. Sectionswere stained with rabbit anti-RAGE IgG. Increased expression of RAGE wasnoted in the podocytes of the glomerulus.

FIG. 2. Incubation of HUVECs with EN-RAGE results in increased cellsurface VCAM-1. Human umbilical vein endothelial cells were cultured inserum-free RPMI 1640 without endothelial cell growth factor for 24 hrsand then stimulated with EN-RAGE or bovine serum albumin (BSA); both 10μg/ml. Where indicated, cells were pretreated with rabbit anti-humanRAGE IgG, nonimmune rabbit IgG; in certain cases, EN-RAGE was pretreatedwith the indicated concentration of soluble RAGE (sRAGE) for 2 hrs priorto stimulation with EN-RAGE. After eight hrs stimulation with EN-RAGE,cells were fixed as described above. Cell surface ELISA employinganti-VCAM-1 IgG was performed. Statistical considerations are shown inthe figure.

FIG. 3. Incubation of HUVECs with EN-RAGE increases VCAM-1 functionalactivity: increased binding of Molt-4 cells. Assessment of functionalVCAM-1 activity was determined using ⁵¹Cr-labelled Molt-4 cells (ATCC)as described above. HUVEC were treated with either BSA (10 μg/ml) orEN-RAGE (5 μg/ml) for eight hrs. Molt-4 cells (5×10⁷/ml) were incubatedfor 2 hrs in RPMI containing⁵¹ Cr (0.1 mCi). At the end of that time,cells were washed with PBS and then added to the monolayer of treatedHUVEC for one hour. Unbound Molt-4 cells were removed by washing threetimes with PBS. Cells were then lysed in buffer containing triton-X 100(2%) in order to release Molt-4 cell-bearing radioactivity. Statisticalconsiderations are shown in the figure.

FIG. 4. Delayed hypersensitivity model: suppression of inflammation inthe presence of soluble RAGE. CF-1 mice were sensitized with mBSA; afterthree weeks, mBSA was injected into the hind foot pad. Certain mice weretreated with the indicated concentrations of mouse serum albumin, sRAGEor the indicated F(ab′)₂ antibody fragments of RAGE or EN-RAGE.Inflammation score was defined as above (scale; 1–9).

FIG. 5. Nucleic Acid Sequence of bovine EN-RAGE. The cDNA for bovineEN-RAGE was cloned and deposited with Genbank at Accession No. AF011757. The sequence (5′ to 3′) is shown in FIG. 5 (SEQ ID NO. 1).

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used herein: CML—carboxymethyl-lysine;AGE—advanced glycation endproduct(s); RAGE—receptor for advancedglycation endprocut (s); sRAGE—soluble receptor for advanced glycationendproduct(s); EN-RAGE—Extracellular Novel RAGE Binding Protein.

The present invention provides for an isolated human EN-RAGE peptide. Inone embodiment, the isolated EN-RAGE peptide having the N-terminal aminoacid sequence shown in Table 1. In another embodiment, the EN-RAGEpeptide is encoded by the cDNA sequence of Genbank Accession No. AF 011757. An isolated nucleic acid molecule encoding an EN-RAGE peptide. Inone embodiment, the EN-RAGE peptide is human EN-RAGE. In anotherembodiment, the nucleic acid is DNA, cDNA or RNA. In one example, thenucleic acid sequence of the EN-RAGE is the sequence shown in FIG. 5(Seq I.D. No. 1).

The present invention also provides for a replicable vector comprisingthe EN-RAGE nucleic acid molecule. In one embodiment, the replicablevector is a prokaryotic expression vector, a yeast expression vector, abaculovirus expression vector, or a mammalian expression vector.

The present invention also provides for a host cell comprising thereplicable vector. In one embodiment, the host cell is a eukaryoticcell, a somatic cell, or a germ cell.

In another embodiment, the nucleic acid molecule of the invention may belabelled with a detectable moiety. The detectable moiety may be selectedfrom the group consisting of: a fluorescent label, a digoxigenin, abiotin, an enzyme, a radioactive atom, a paramagnetic ion, and achemiluminescent label.

The present invention also provides for nucleic acid molecule consistingessentially of a unique fragment of an EN-RAGE nucleic acid sequence ina 3′ to 5′ orientation, wherein the sequence antisense to at least aportion of a gene encoding naturally occurring EN-RAGE peptide.

The present invention also provides a composition comprising an EN-RAGEpeptide or fragment thereof and a pharmaceutically acceptable carrier.In one embodiment, the pharmaceutically acceptable carrier is anaerosol, intravenous, oral or topical carrier.

The present invention also provides for an antibody immunoreactive withan epitope comprising a unique sequence of EN-RAGE.

The present invention also provides for a ribozyme which is capable ofspecifically cleaving EN-RAGE mRNA in a cell.

The present invention also provides for a transgenic nonhuman mammalwhose germ or somatic cells contain a nucleic acid molecule whichencodes an EN-RAGE peptide or a biologically active variant thereof,introduced into the mammal, or an ancestor thereof, at an embryonicstage. In one embodiment, the nucleic acid molecule which encodesEN-RAGE polypeptide is overexpressed in the cells of the mammal. Inanother embodiment, the nucleic acid molecule encodes human EN-RAGEpeptide. In another embodiment, the active variant comprises a homologof EN-RAGE.

The present invention also provides for a transgenic nonhuman mammalwhose germ or somatic cells have been transfected with a suitable vectorwith an appropriate sequence designed to reduce expression levels ofEN-RAGE peptide below the expression levels of that of a native mammal.In one embodiment, the suitable vector contains an appropriate piece ofcloned genomic nucleic acid sequence to allow for homologousrecombination. In another emboidment, the suitable vector encodes aribozyme capable of cleaving an EN-RAGE mRNA molecule or an antisensemolecule which comprises a sequence antisense to naturally occurringEN-RAGE mRNA sequence.

The present invention also provides for a method for determining whethera compound is capable of inhibiting the interaction of an EN-RAGEpeptide with a RAGE peptide, which comprises: (a) admixing: (i) a RAGEpeptide or an sRAGE peptide or a fragment of either thereof, (ii) anEN-RAGE peptide or a fragment thereof, and (iii) the compound; (b)measuring the level of interaction between the peptide of step (a) (i)and the peptide of step (a) (ii), and (c) comparing the amount ofinteraction meausred in step (b) with the amount measured between thepeptide of step (a) (i) and the peptide of step (a) (ii) in the absenceof the compound, thereby determining whether the compound is capable ofinhibiting the interaction of the EN-RAGE peptide with the RAGE peptide,wherein a reduction in the amount of interaction in the presence of thecompound indicates that the compound is capable of inhibiting theinteraction.

In one embodiment, the fragment of step (a) (i) is the V-domain of RAGE.In another embodiment, the fragment of step (a) (i) or (a)(ii) issynthetic. In another embodiment, the compound comprises at least aportion of naturally occuring sRAGE peptide. In another embodiment, thecompound is a peptidomimetic. In another embodiment, the compound is anorganic molecule. In another embodiment, the compound is a peptide, anucleic acid or an inorganic chemical. In another embodiment, thecompound is a molecule of less than 10,000 daltons. In anotherembodiment, the compound is an antibody or fragment thereof. In anotherembodiment, the compound is a mutated RAGE peptide or a fragmentthereof. In another embodiment, the compound is a mutated sRAGE peptideor a fragment thereof. In another embodiment, the compound is a mutatedEN-RAGE peptide or a fragment thereof. In another embodiment, thepeptide of step (a) (i) is affixed to a solid surface. In anotherembodiment, the peptide of step (a)(ii) is affixed to a solid surface.In another embodiment, the peptide of step (a) (i) or (a) (ii) isdetectably labeled. In another embodiment, the detectable labelcomprises fluorescence, biotin, or radioactivity.

In another embodiment, the admixing in the screening method occurs in acell. In another embodiment, the admixing occurs in an animal.

The present invention also provides for a compound identified by thescreening method described herein which compound is useful for thesuppression of inflammation in a subject.

The present invention also provides for a compound identified by themethod described herein which is useful for the treatment of systemiclupus erythematosus or inflammatory lupus nephritis in a subject.

The present invention provides for a previously unknown compoundidentified by the method described hereinabove.

The present invention also provides for a method for inhibitinginflammation in a subject which comprises administering to the subject acompound capable of interfering with the interaction between EN-RAGEpeptide and receptor for advanced glycation endproduct (RAGE) in thesubject thereby inhibiting inflammation in the subject.

In another embodiment, the compound is an anti-EN-RAGE antibody or afragment thereof or an anti-RAGE antibody or fragment thereof. Inanother embodiment, the compound is an sRAGE peptide. In anotherembodiment, the compound consists essentially of the ligand bindingdomain of sRAGE peptide or the ligand binding domain of EN-RAGE peptide.In another embodiment, the compound is a nucleic acid molecule or apeptide. In another embodiment, the peptide is an antibody or a fragmentthereof. In another embodiment, the nucleic acid molecule is a ribozymeor an antisense nucleic acid molecule. In another embodiment, thecompound is a compound identified by the screening method describedhereinabove.

In another embodiment, the inflammation is assoicated with delayedhypersensitivity, accelerated athrosclerosis, or lupus nephritis. Inanother embodiment, the subject is a human, a primate, a mouse, a rat ora dog.

In another embodiment, the administration comprises intralesional,intraperitoneal, intramuscular or intravenous injection; infusion;liposome-mediated delivery; or topical, intrathecal, gingival pocket,per rectum, intrabronchial, nasal, oral, ocular or otic delivery. Inanother embodiment, the compound is administered hourly, daily, weekly,monthly or annually. In another embodiment, the effective amount of thecompound comprises from about 0.000001 mg/kg body weight to about 100mg/kg body weight.

In another embodiment, the subject is suffering from systemic lapuserythematosus, inflammatory lupus nephritis, septic shock orendotoxemia. In another embodiment, the subject is suffering frominflammation.

In a further embodiment, the subject is suffering from an autoimmune orinflammatory disorder in which recruitment of EN-RAGE-containinginflammatory cells occurs. In another embodiment, the subject issuffering from a bacterial-associated or other pathogen-associatedinfection.

In another embodiment, the method further comprises administering to thesubject a pharmaceutically acceptable carrier during the administrationof the compound. In another embodiment the carrier comprises a diluent.In another embodiment, the carrier comprises, a virus, a liposome, amicroencapsule, a polymer encapsulated cell or a retroviral vector. Inanother embodiment, the carrier is an aerosol, intravenous, oral ortopical carrier. In another embodiment, the compound is administeredfrom a time release implant.

The present invention also provides for a method for determining whethera compound is capable of inhibiting the ability of EN-RAGE protein tobind with a second protein which comprises: (a) admixing the EN-RAGEprotein, the second protein and the compound; (b) measuring the amountof binding between the EN-RAGE protein and the second protein; and (c)comparing the amount of binding measured in step (b) with the amount ofbinding between EN-RAGE and the second protein in the absence of thecompound, wherein a reduction in the amount of binding indicates thatthe compound is capable of inhibiting the ability of EN-RAGE protein tobind with the second protein.

The human cDNA of RAGE is 1406 base pairs and encodes a mature proteinof 404 amino acids. See FIG. 3 of Neeper et al. 1992. As used herein,“V-domain of RAGE” refers to the immunoglobulin-like variable domain asshown in FIG. 5 of Neeper, M., Schmidt, A. M., Brett, J., Yan, S. D.,Wang, F., Pan, Y. C., Elliston, K., Stern, D., and Shaw, A. Cloning andexpression of RAGE: a cell surface receptor for advanced glycosylationend products of proteins. J. Biol. Chem. 267:14998–15004, 1992 thecontents of which are hereby incorporated by reference. The V-domainincludes amino acids from position 23 to position 120 as shown in FIG. 4of Neeper et al. (1992). The leader sequence shown is not part of theV-domain and in the human, t domain begins with the amino acidsA-Q-N-I-T(SEQ ID NO: 5) . . . . The minimum required amino acid sequenceto define the AGE binding site in the RAGE protein may be much smallerthan 120 amino acids.

The bovine EN-RAGE nucleic acid sequence has been cloned and has beendeposited with Genbank at Accession No. AF 011757. The nucleic acidsequence of EN-RAGE is shown in FIG. 5. Homologs of EN-RAGE present inother species would be obtainable via methods known to one of skill inthe art. For example, sequences unique to the bovine EN-RAGE nucleicacid cDNA sequence may be used as probes to screen a human cDNA libraryin order to obtain the human homolog.

Ligands for RAGE such as AGEs (CML-modified AGEs) and p12, aproinflammatory cytokine, activate inflammatory cells. This has beenshown in mice. These activation effects are blocked in the presence ofsRAGE. Thus, the present invention provides methods for blockinginflammation (e.g., inflammation due to immune stimulation) in a subjectby administering a compound which is capable of interfering with theinteraction between EN-RAGE and RAGE in a subject. Such a method wouldbe selective for inflammation. The compound, in one example, is designedspecifically as a competitive inhibitor of ligands for RAGE.

The screening assay may be carried out wherein one of the components isbound or affixed to a solid surface. In one embodiment the peptide isaffixed to a solid surface. In another embodiment, the second peptidewhich has the sequence of the AGE binding site of RAGE is bound oraffixed to a solid surface. The solid surfaces useful in this embodimentwould be known to one of skill in the art. For example, one embodimentof a solid surface is a bead, a column, a plastic dish, a plastic plate,a microscope slide, a nylon membrane, etc. The material of which thesolid surface is comprised is synthetic in one example.

One of the components of step (a) of the screening assay may bedetectably labelled. The component (either the compound, the peptide orthe V-domain or second peptide) may be labeled with a detectable moietyincluding a fluorescent label, a biotin, a digoxigenin, a radioactiveatom, a paramagnetic ion, and a chemiluminescent label. The componentmay be labeled by covalent means such as chemical, enzymatic or otherappropriate means with a moiety such as an enzyme or radioisotope.

In one embodiment, the subject is be a mammal. In another embodiment,the subject is a vertebrate. In a preferred embodiment, the mammal is ahuman. In one example, the subject is a diabetic subject. In anotherexample of the invention, the subject is suffering from diabetes, renalfailure, amyloidoses, aging or inflammation. The subject may be an obesesubject as defined by the American Medical Association height and weightstandards. The subject may be aged. The subject may be a human, aprimate, an equine subject, an opine subject, an avian subject, a bovinesubject, a porcine, a canine, a feline or a murine subject.

In one embodiment, the subject is suffering from an AGE-related disease.In another embodiment, such AGE-related disease is manifest in thebrain, retina, kidney, vasculature, heart, or lung. In anotherembodiment, the subject is suffering from Alzheimer's disease or adisease which is manifested by AGEs accumulating in the subject. Inanother embodiment, the subject is suffering from symptoms of diabetessuch as soft tissue injury, reduced ability to see, cardiovasculardisease, kidney disease, etc. Such symptoms would be known to one ofskill in the art.

The compound may be a polypeptide. The polypeptide may be a peptide; apeptidomimetic, a synthetic polypeptide, a derivative of a naturalpolypeptide, a modified polypeptide, a labelled polypeptide, or apolypeptide which includes non-natural peptides. The peptidomimetic maybe identified from screening large libraries of different compoundswhich are peptidomimetics to determine a compound which is capable ofpreventing accelerated atherosclerosis in a subject predisposed thereto.The polypeptide may be a non-natural polypeptide which has chirality notfound in nature, i.e. D-amino acids or L-amino acids.

In one embodiment, the compound is an antagonist, wherein the antagonistis capable of binding the RAGE with higher affinity than AGEs, thuscompeting away the effects of AGE's binding.

In another embodiment, the compound may be a ribozyme which is capableof inhibiting expression of RAGE. In another embodiment, the compound isan anti-RAGE antibody, an anti-AGE antibody, an anti-V-domain of RAGEantibody. The antibody may be monoclonal, polyclonal, chimeric,humanized, primatized. The compound may be a fragment of such antibody.

In another embodiment of the present invention, the method may furthercomprise administering to the subject a pharmaceutically acceptablecarrier during the administration of the polypeptide. The administrationmay comprise intralesional, intraperitoneal, intramuscular orintravenous injection; infusion; liposome-mediated delivery; or topical,nasal, oral, ocular or otic delivery. In a further embodiment, theadministration includes intrabronchial administration, anal orintrathecal administration.

The polypeptide may be delivered hourly, daily, weekly, monthly, yearly(e.g. in a time release form) or as a one time delivery. The deliverymay be continuous delivery for a period of time, e.g. intravenousdelivery.

The effective amount of the polypeptide may comprise from about 0.000001mg/kg body weight to about 100 mg/kg body weight. In one embodiment, theeffective amount may comprise from about 0.001 mg/kg body weight toabout 50 mg/kg body weight. In another embodiment, the effective amountmay range from about 0.01 mg/kg body weight to about 10 mg/kg bodyweight. The actual effective amount will be based upon the size of thepolypeptide, the biodegradability of the polypeptide, the bioactivity ofthe polypeptide and the bioavailability of the polypeptide. If thepolypeptide does not degrade quickly, is bioavailable and highly active,a smaller amount will be required to be effective. The effective amountwill be known to one of skill in the art; it will also be dependent uponthe form of the polypeptide, the size of the polypeptide and thebioactivity of the polypeptide. One of skill in the art could routinelyperform empirical activity tests for a polypeptide to determine thebioactivity in bioassays and thus determine the effective amount.

In another embodiment of the present invention, the method may furthercomprise administering a pharmaceutically acceptable carrier to thesubject during the administration of the compound. The administrationmay comprise intralesional, intraperitoneal, intramuscular orintravenous injection; infusion; liposome-mediated delivery; or topical,nasal, oral, ocular or otic delivery.

The compound may be administered hourly, daily, weekly, monthly, yearly(e.g. in a time release form) or as a one time delivery. The delivery oradministration may be continuous delivery for a period of time, e.g.intravenous delivery.

The compound may be an sRAGE polypeptide such as polypeptide analogs ofsRAGE. Such analogs include fragments of sRAGE. Following the proceduresof the published application by Alton et al. (WO 83/04053), one canreadily design and manufacture genes coding for microbial expression ofpolypeptides having primary conformations which differ from that hereinspecified for in terms of the identity or location of one or moreresidues (e.g., substitutions, terminal and intermediate additions anddeletions). Alternately, modifications of cDNA and genomic genes can bereadily accomplished by well-known site-directed mutagenesis techniquesand employed to generate analogs and derivatives of sRAGE polypeptide.Such products share at least one of the biological properties of sRAGEbut may differ in others. As examples, products of the invention includethose which are foreshortened by e.g., deletions; or those which aremore stable to hydrolysis (and, therefore, may have more pronounced orlongerlasting effects than naturally-occurring); or which have beenaltered to delete or to add one or more potential sites forO-glycosylation and/or N-glycosylation or which have one or morecysteine residues deleted or replaced by e.g., alanine or serineresidues and are potentially more easily isolated in active form frommicrobial systems; or which have one or more tyrosine residues replacedby phenylalanine and bind more or less readily to target proteins or toreceptors on target cells. Also comprehended are polypeptide fragmentsduplicating only a part of the continuous amino acid sequence orsecondary conformations within sRAGE, which fragments may possess oneproperty of sRAGE and not others. It is noteworthy that activity is notnecessary for any one or more of the polypeptides of the invention tohave therapeutic utility or utility in other contexts, such as in assaysof sRAGE antagonism. Competitive antagonists may be quite useful in, forexample, cases of overproduction of sRAGE.

Of applicability to polypeptide analogs of the invention are reports ofthe immunological property of synthetic peptides which substantiallyduplicate the amino acid sequence extant in naturally-occurringproteins, glycoproteins and nucleoproteins. More specifically,relatively low molecular weight polypeptides have been shown toparticipate in immune reactions which are similar in duration and extentto the immune reactions of physiologically-significant proteins such asviral antigens, polypeptide hormones, and the like. Included among theimmune reactions of such polypeptides is the provocation of theformation of specific antibodies in immunologically-active animals[Lerner et al., Cell, 23, 309–310 (1981); Ross et al., Nature, 294,654–658 (1981); Walter et al., Proc. Natl. Acad. Sci. USA, 78, 4882–4886(1981); Wong et al., Proc. Natl. Sci. USA, 79, 5322–5326 (1982); Baronet al., Cell, 28, 395–404 (1982); Dressman et al., Nature, 295, 185–160(1982); and Lerner, Scientific American, 248, 66–74 (1983). See also,Kaiser et al. [Science, 223, 249–255 (1984)] relating to biological andimmunological properties of synthetic peptides which approximately sharesecondary structures of peptide hormones but may not share their primarystructural conformation.

The compound of the present invention may be a peptidomimetic compoundwhich may be at least partially unnatural. The peptidomimetic compoundmay be a small molecule mimic of a portion of the amino acid sequence ofsRAGE. The compound may have increased stability, efficacy, potency andbioavailability by virtue of the mimic. Further, the compound may havedecreased toxicity. The peptidomimetic compound may have enhancedmucosal intestinal permeability. The compound may be syntheticallyprepared. The compound of the present invention may include L-,D- orunnatural amino acids, alpha, alpha-disubstituted amino acids, N-alkylamino acids, lactic acid (an isoelectronic analog of alanine). Thepeptide backbone of the compound may have at least one bond replacedwith PSI—[CH═CH] (Kempf et al. 1991). The compound may further includetrifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine,poly-L-propargylglycine, poly-D,L-allyl glycine, or poly-L-allylglycine.

One embodiment of the present invention is a peptidomimetic compoundwherein the compound has a bond, a peptide backbone or an amino acidcomponent replaced with a suitable mimic. Examples of unnatural aminoacids which may be suitable amino acid mimics include β-alanine,L-α-amino butyric acid, L-γ-amino butyric acid, L-α-amino isobutyricacid, L-ε-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid,L-glutamic acid, cysteine (acetamindomethyl), N-ε-Boc-N-α-CBZ-L-lysine,N-ε-Boc-N-α-Fmoc-L-lysine, L-methionine sulfone, L-norleucine,L-norvaline, N-ε-Boc-N-δCBZ-L-ornithine, N-δ-Boc-N-α-CBZ-L-ornithine,Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, Boc-L-thioproline.(Blondelle, et al. 1994; Pinilla, et al. 1995).

In another embodiment, the compound may be soluble RAGE (sRAGE) or afragment thereof. Soluble RAGE is not located on the cell surface and isnot associated with a cell membrane.

The subject may be a mammal or non-mammal. The subject may be a human.The subject may be a mouse, a rat, a cow, a monkey, a horse, a pig, or adog. The subject may be a diabetic subject.

The administration of the compound may be intralesional,intraperitoneal, intramuscular or intravenous injection; infusion;liposome-mediated delivery; topical, nasal, oral, anal, ocular or oticdelivery. The administration may be constant for a certain period oftime or periodic and at specific intervals. The carrier may be adiluent, an aerosol, a topical carrier, an aqueous solution, anonaqueous solution or a solid carrier.

In the practice of any of the methods of the invention or preparation ofany of the pharmaceutical compositions a “therapeutically effectiveamount” is an amount which is capable of preventing interaction ofEN-RAGE/RAGE in a subject. Accordingly, the effective amount will varywith the subject being treated, as well as the condition to be treated.For the purposes of this invention, the methods of administration are toinclude, but are not limited to, administration cutaneously,subcutaneously, intravenously, parenterally, orally, topically, or byaerosol.

As used herein, the term “suitable pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers, suchas phosphate buffered saline solution, water, emulsions such as anoil/water emulsion or a triglyceride emulsion, various types of wettingagents, tablets, coated tablets and capsules. An example of anacceptable triglyceride emulsion useful in intravenous andintraperitoneal administration of the compounds is the triglycerideemulsion commercially known as Intralipid®.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients.

This invention also provides for pharmaceutical compositions includingtherapeutically effective amounts of polypeptide compositions andcompounds, together with suitable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions may be liquidsor lyophilized or otherwise dried formulations and include diluents ofvarious buffer content (e.g., Tris-HCl., acetate, phosphate), pH andionic strength, additives such as albumin or gelatin to preventabsorption to surfaces, detergents (e.g., Tween 20, Tween 80, PluronicF68, bile acid salts), solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulkingsubstances or tonicity modifiers (e.g., lactose, mannitol), covalentattachment of polymers such as polyethylene glycol to the compound,complexation with metal ions, or incorporation of the compound into oronto particulate preparations of polymeric compounds such as polylacticacid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multi lamellar vesicles, erythrocyteghosts, or spheroplasts.

Such compositions will influence the physical state, solubility,stability, rate of in vivo release, and rate of in vivo clearance of thecompound or composition. The choice of compositions will depend on thephysical and chemical properties of the compound.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended bythe invention are particulate compositions coated with polymers (e.g.,poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

When administered, compounds are often cleared rapidly from thecirculation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of bioactive compounds may by required to sustaintherapeutic efficacy. Compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline areknown to exhibit substantially longer half-lives in blood followingintravenous injection than do the corresponding unmodified compounds(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987).Such modifications may also increase the compound's solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound adducts less frequently or in lower doses thanwith the unmodified compound.

Attachment of polyethylene glycol (PEG) to compounds is particularlyuseful because PEG has very low toxicity in mammals (Carpenter et al.,1971). For example, a PEG adduct of adenosine deaminase was approved inthe United States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. A second advantage afforded by theconjugation of PEG is that of effectively reducing the immunogenicityand antigenicity of heterologous compounds. For example, a PEG adduct ofa human protein might be useful for the treatment of disease in othermammalian species without the risk of triggering a severe immuneresponse. The polypeptide or composition of the present invention may bedelivered in a microencapsulation device so as to reduce or prevent anhost immune response against the polypeptide or against cells which mayproduce the polypeptide. The polypeptide or composition of the presentinvention may also be delivered microencapsulated in a membrane, such asa liposome.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxy-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate group inproteins.

Pharmaceutical with Carriers

In one preferred embodiment the pharmaceutical carrier may be a liquidand the pharmaceutical composition would be in the form of a solution.In another equally preferred embodiment, the pharmaceutically acceptablecarrier is a solid and the composition is in the form of a powder ortablet. In a further embodiment, the pharmaceutical carrier is a gel andthe composition is in the form of a suppository or cream. In a furtherembodiment the active ingredient may be formulated as a part of apharmaceutically acceptable transdermal patch.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by for example, intramuscular, intrathecal,epidural, intraperitoneal or subcutaneous injection. Sterile solutionscan also be administered intravenously. The active ingredient may beprepared as a sterile solid composition which may be dissolved orsuspended at the time of administration using sterile water, saline, orother appropriate sterile injectable medium. Carriers are intended toinclude necessary and inert binders, suspending agents, lubricants,flavorants, sweeteners, preservatives, dyes, and coatings.

The active ingredient of the present invention (i.e., the compoundidentified by the screening method or composition thereof) can beadministered orally in the form of a sterile solution or suspension oncontaining other solutes or suspending agents, for example, enoughsaline or glucose to make the solution isotonic, bile salts, acacia,gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitoland its anhydrides copolymerized with ethylene oxide) and the like.

The active ingredient can also be administered orally either in liquidor solid composition form. Compositions suitable for oral administrationinclude solid forms, such as pills, capsules, granules, tablets, andpowders, and liquid forms, such as solutions, syrups, elixirs, andsuspensions. Forms useful for parenteral administration include sterilesolutions, emulsions, and suspensions.

In another embodiment of the present invention, the subject may havediabetes. The subject may demonstrate complications associated withdiabetes. Some examples of such complications include activation ofendothelial and macrophage AGE receptors, altered lipoproteins, matrix,and basement membrane proteins; altered contractility and hormoneresponsiveness of vascular smooth muscle; altered endothelial cellpermeability; sorbitol accumulation; neural myoinositol depletion oraltered Na—K ATPase activity. Such complications are discussed in arecent publication by Porte and Schwartz, Diabetes Complications: Why isGlucose potentially Toxic?, Science, Vol. 272, pages 699–700.

This invention is illustrated in the Experimental Details section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS

The present invention provides for a new proinflammatory cytokine-likemolecule (EN-RAGE) (which has some sequence similarity to the family ofcalgranulin molecules). EN-RAGE is a protein located inside ofinflammatory cells (such as neutrophils) and which may be released bysuch inflammatory cells. EN-RAGE has biological activity that may beresponsible for the propagation and sustainment of an inflammatoryresponse by interacting with cellular receptor RAGE.

Example 1 Interaction of EN-RAGE (Extracellular Novel Rage BindingProtein) with Receptor for AGE (RAGE) Perpetuates InflammatoryResponses: Suppression of Delayed-Type Hypersensitivity Reactions withSoluble Receptor for Age (sRAGE)

Expression of RAGE, the Receptor for Advanced Glycation Endproducts, isincreased in the setting of inflammation. Here we report a new member ofthe calgranulin family of proinflammatory cytokines called EN-RAGE (orExtracellular Novel RAGE-binding protein), which interacts with RAGE oncells such as endothelial cells, to alter cellular properties in amanner consistent with perturbation. Administration of soluble RAGE (theextracellular ligand binding domain of RAGE; sRAGE) or anti-RAGE oranti-EN-RAGE F(ab′)₂ fragments markedly attentuated inflammation in amodel of delayed hypersensitivity. These data link RAGE to theinflammatory response and identify EN-RAGE and RAGE as novel targets foranti-inflammatory intervention. Soluble RAGE, furthermore, is thus aprototypic structure for the design of a new class of anti-inflammatoryagents.

The Receptor for AGE (RAGE) is a member of the immunoglobulinsuperfamily of cell-surface molecules (1–2). Originally identified andcharacterized as a cellular receptor for glucose (aldose sugar)-modifiedproteins, or Advanced Glycation Endproducts (AGEs) (3–13), RAGE hassubsequently been reported to interact with other ligands, in bothsettings of normal development and in Alzheimer's disease (14–16). Innormal development, RAGE interacts with amphoterin, a polypeptide whichmediates neurite outgrowth in cultured embryonic neurons. In thosestudies, either anti-RAGE F(ab′)₂ or soluble RAGE (sRAGE) inhibitedneurite outgrowth on amphoterin-coated matrices, but not on matricescoated with other substrates such as laminin or poly-l-lysine (3). Inlater studies, RAGE was identified as a receptor on neurons andmicroglia for amyloid-β-peptide, a polypeptide linked to thepathogenesis of neuronal toxicity and death in Alzheimer's disease.

In unpublished observations from our laboratory, we identified thatincreased RAGE expression was noted in the vascular and inflammatorycells of inflammatory lesions, such as in the kidney tissue frompatients with active lupus nephritis (FIG. 1). We therefore hypothesizedthat RAGE might interact with alternative ligand(s) in that setting inorder to, perhaps, participate in the inflammatory response.

Herein, the findings demonstrate that RAGE interacts with a moleculewith close homology to calgranulin C. We have termed this molecule,EN-RAGE (Extracellular Novel RAGE binding protein) and show thatEN-RAGE:RAGE interaction activates cells such as endothelial cells whichare importantly involved in the inflammatory response. In a model ofmurine delayed hypersensitivity, administration of soluble RAGE (sRAGE),which contains the ligand interaction domain, inhibits the developmentof cellular activation and inflammation. These findings identify RAGE asa new target for anti-inflammatory intervention.

Materials and Methods

Isolation and Purification of EN-RAGE. Bovine lung acetone powder(SIGMA®) was subjected to solubilization in buffer containing tris(0.02M, pH 7.4); NaCl (0.15 M); octyl-β-glucoside (1%); and proteaseinhibitors (PMSF and aprotinin). After serial chromatography onto SPsepharose (Pharmacia LKB®), and affi-gel 10 resin (BIO-RAD®) to whichhad been adsorbed purified soluble human RAGE (prepared from abaculovirus expression system), RAGE-binding proteins were identifiedbased on a screening assay employing immobilized column fraction (NuncMaxisorp dishes) (NUNC®) and ¹²⁵-I-labelled sRAGE as above. Afterelution with heparin-containing buffer (1 mg/ml), positive fractionswere identified. RAGE-binding proteins were subjected to sequenceanalysis.

Cloning of EN-RAGE. The cDNA for EN-RAGE was cloned from a bovine lunglibrary and placed into a baculovirus expression system. In this system,EN-RAGE, which lacks a leader sequence, was synthesized within Sf9cells. EN-RAGE was then purified after solubilization of the cells indetergent-containing buffer, and sequential purification onhydroxylapatite and heparin-containing resins. The final productdisplayed a single band on Coomassie-stained SDS-PAGE gels and wasdevoid of endotoxin after chromatography onto Detoxi-gel columns(PIERCE®). Absence of detectable endotoxin was confirmed using limulusamebocyte assay (SIGMA®).

Sequence analysis. After SDS-PAGE identified an ≈12 kDa polypeptide withRAGE-binding activity, the gel band was eluted according topreviously-published methods (17). The published method was modified byaddition of a final wash of two aliquots (0.1 ml each) of guanidine(5.0M), urea (5.0M), trifluoroacetic acid (0.2%), acetonitrile (10%),and Zwittergent 3-08 (1.0%) (Calbiochem) to ensure that protein wascompletely washed from the filter. Amino-terminal sequence analysis wasperformed. Automated Edman degradation was carried out employing anHP-G1005A sequencer (Hewlett Packard Analytical Instruments). In orderto obtain internal sequence, the gel bands were treated as above forelution, except that the extraction buffer contained half the usualamount of SDS (1). Endoproteinase Lys-C (1 μg) (Boehringer Mannheim) wasadded and the sample incubated overnight. The digest was thenfractionated by microbore HPLC (Michrom Bioresources) on a 1 mm×50 mmPLRP—S column (Polymer Laboratories, Ltd.). The gradient utilized was 2%per minute from acetonitrile (5–75%) in trifluoroacetic acid (0.1%) andfractions were collected at 30 second intervals. Absorbance wasmonitored at 214 nm and fractions that corresponded to chromatographicpeaks were then subjected to sequence analysis.

Endothelial cell activation. Human umbilical vein endothelial cells wereisolated, characterized and maintained as previously described (18).Cells were cultured in serum-free RPMI 1640 without endothelial cellgrowth factor for 24 hrs and then stimulated with the indicatedconcentrations of EN-RAGE. Where indicated, cells were pretreated withrabbit anti-human RAGE IgG, nonimmune rabbit IgG; in certain cases,EN-RAGE was pretreated with the indicated concentration of soluble RAGE(sRAGE) for 2 hrs prior to stimulation with EN-RAGE. After eight hrsstimulation with EN-RAGE, cells were fixed with paraformaldehyde (2%)for 30 mins, washed twice with PBS, treated with PBS containing non-fatdry milk (5%) and BSA (2.5%) to block nonspecific binding sites on thecell surface. Cell surface ELISA employing anti-VCAM-1 IgG (Santa CruzBiotechnologies, Santa Cruz, Calif.) was performed. Assessment offunctional VCAM-1 activity was determined using ⁵¹Cr-labelled Molt-4cells (ATCC) as previously described (10).

Delayed hypersensitivity model. A murine model of delayedhypersensitivity was established based on previously-published studies(19). Female CF-1 mice (Charles River laboratories), 6 weeks of age,were sensitized by subcutaneous injection over the left inguinal lymphnode of an emulsion (0.1 ml) containing methylated BSA (mBSA; 25 mg/ml;SIGMA®), NaCl (0.9%), dextran (5–40×10⁶ MW; 50 mg/ml; SIGMA®) andFreund's incomplete adjuvant (50%; ICN Biomedical). Three weeks later,the left plantar hind paw was injected subcutaneously with mBSA (0.4mg/ml; 0.050 ml). Where indicated, mice were pretreated byintraperitoneal injection with sRAGE (indicated dose), mouse serumalbumin (SIGMA®), immune or nonimmune F(ab′)₂ fragments (prepared usinga kit from Pierce) 24 and 12 hrs prior to, and 6 and 12 hrs after localchallenge with MBSA. 24 hrs after injection of foot pad with mBSA,clinical score of foot pad was performed; mice were then humanelysacrificed and feet fixed in formalin (10%) or frozen for furtheranalysis. Histologic score was performed on sections of foot stainedwith hematoxylin and eosin (SIGMA®). The clinical score was defined asfollows (scale; 1–5): 1=no inflammation and thus identical to untreatedfoot; 2=slight rubor and edema; 3=severe rubor and edema with wrinklingof the skin of the foot pad; 4=severe rubor and edema without wrinklingof the skin of the foot pad; and 5=severe rubor and edema resulting inspreading of the toes. The histologic score after hematoxylin and eosinstaining was defined as follows (scale; 1–5): 1=no leukocyticinfiltration with slight subcutaneous edema; 2=slight perivascularleukocytic infiltration with slight subcutaneous edema; 3=severeleukocytic infiltration without granulomata; and 4=severe leukocyticinfiltration with granulomata.

Results

Identification of EN-RAGE. After a serial series of experiments designedto identify RAGE-binding proteins from bovine lung extract (from whereRAGE was originally purified), an ≈12 kDa polypeptide was identified.Upon sequence analysis, this polypeptide was found to bear significanthomology to members of the calgranulin C family of proteins (Table 1)(20–21). This class of proteins exist intracellularly withininflammatory cells. Upon release in inflamed loci, we postulated theymight be able to, in turn, engage and activate other cells alreadyrecruited into the inflammatory response. Thus, this might represent animportant means by which the inflammatory response might be propagatedand sustained, thereby increasing the probability of cellular injury.

EN-RAGE activates endothelial cells in a RAGE-dependent manner. To testthis hypothesis, EN-RAGE was purified as described above and incubatedwith endothelial cells. Incubation of EN-RAGE with HUVEC resulted inincreased cell surface Vascular Cell Adhesion Molecule-1 (VCAM-1) in aRAGE-dependent manner (FIG. 2). These data suggested that in aninflammatory focus, interaction of EN-RAGE with EC RAGE might representa means by which to further propagate an inflammatory response.Consistent with increased VCAM-1 antigen on the surface ofEN-RAGE-treated ECs, increased binding for Molt-4 cells (which bear theligand for VCAM-1, VLA-4), ensued (FIG. 3). While incubation with eitherBSA or non-immune IgG did not affect the ability of EN-RAGE to activateEC VCAM-1, incubation with either sRAGE or anti-RAGE F(ab′)₂significantly attenuated the ability of EN-RAGE to increase Molt-4binding to treated HUVEC.

We sought to test these hypotheses in in vivo models. We demonstratedthat in diabetic mice, in which the ligand for RAGE is likely to be, atleast in part, products of glycation/oxidation of proteins/lipids, theAdvanced Glycation Endproducts, or AGEs, administration of the soluble,ligand-binding portion of RAGE (soluble or sRAGE), suppressedaccelerated atherosclerosis in diabetic apolipoprotein E null mice (12)and improved wound healing in genetically-diabetic db+/db+mice (22).Thus, the biologic effects of EN-RAGE in highly-inflammatory foci, suchas those characterized by models of granulomatous inflammatory lesions(delayed hypersensitivity), could be suppressed in the presence ofsRAGE.

To test this, we studied a model of delayed hypersensitivity (DH) inwhich mice were first sensitized by injection of methylated BSA (mBSA;which does not bind RAGE) over the inguinal lymph nodes of female CF-1mice. Three weeks after sensitization, mice were challenged with mBSA byinjection into the hind foot pad. An inflammation score was designed ona scale of 1–9 which included both clinical score (1–4) and histologicscore (1–5) as indicated in FIG. 4.

Consistent with our hypothesis, administration of sRAGE suppressedinflammation upon injection of mBSA into the foot pad of micepreviously-sensitized with mBSA over the lymph nodes, in adose-dependent manner (FIG. 4). At a dose of 100 μg sRAGE, inflammationwas markedly suppressed (p<0.01). In contrast, administration of mouseserum albumin, had no effect on the appearance of the inflammatorylesion (FIG. 4). Consistent with an important role for EN-RAGE and RAGEin the development of inflammation in this model, treatment of the micewith either anti-EN-RAGE F(ab′)₂ or anti-RAGE F(ab′)₂ considerablysuppressed inflammation (p<0.05 in each case compared with treatmentwith nonimmune F(ab′)₂. When mice were treated with both anti-EN-RAGEand anti-RAGE F(ab′)₂, even further suppression of the inflammatoryresponse eventuated (p<0.05 compared with treatment with nonimmuneF(ab′)₂ (FIG. 4).

DISCUSSION

The inflammation phenotype observed in delayed-type hypersensitivityreactions certainly represent the culmination of a complex interplay andcontribution of multiple cell types and their cellular mediators. In thedevelopment of inflammation, an important source of the stimuli may befrom the inflammatory cells themselves. Upon initial recruitment into aninflammatory locus, cells such as neutrophils and macrophages mayrelease mediators such as those of the calgranulin family, includingEN-RAGE, and propagate and sustain the inflammatory response. Suchmediators, such as EN-RAGE, likely require cellular receptors toinitiate events that will culminate in altered gene expression.

Our data strongly suggest that EN-RAGE-RAGE interaction is an importantfactor in these processes. Nearly complete suppression of inflammationwas noted in the presence of sRAGE, in a dose-dependent manner. Basedupon our studies, sRAGE may act as a decoy in this setting to bindEN-RAGE prior to its ability to engage RAGE-bearing cells implicated inthe inflammatory response. Furthermore, in the presence ofanti-RAGE/anti-EN-RAGE or anti-RAGE+anti-EN-RAGE F(ab′)₂, substantialsuppression of inflammation was observed, further indicating a role ofthese factors in the modulation of the inflammatory response.

It is important to note, of course, that alternate mechanisms underlyingthe beneficial effects of sRAGE may be operative in these settings.However, the studies noted above employing the indicated F(ab′)₂fragments, strongly implicate EN-RAGE and RAGE in the evolution of theinflammatory response in this setting.

In conclusion, the studies presented herein implicate RAGE centrally inthe inflammatory response and identify soluble RAGE as a prototypicstructure for the development of novel, anti-inflammatory agents.

Note: FIG. 5 shows the nucleic acid sequence (cDNA sequence) of bovineEN-RAGE.

TABLE 1 Sequence analysis of EN-RAGE and comparison with relatedproteins.           10       20       30 EN-RAGE (SEQ ID NO:2)  TKLEDHLEGIINIGHQYSVRVGHFDTLNKY - N-TERM Endo Lys C (SEQ ID NO.5)B-COAg (SEQ ID NO.3)   TKLEDHLEGIINIFHQYSVRVGHFDTLNKR B-CAAF1 (SEQ IDNO.4)   TKLEDHLEGIINIFHQYSVRVGHFDTLNKR  31      40           50      60EN-RAGE   ELKQLGTKELPKTLQNXKDQ N-TERM Endo Lys C B-COAg  ELKQLITKELPKTLQNTKDQPTIDKIFQDL B-CAAF1  ELKQLITKELPKTLQNTKDQPTIDKIFQDL 61       70       80       90 EN-RAGEN-TERM Endo Lys C     DGAVSFEEFVVLVSRVLK B-COAg DADKDGAVSFEEFVVLVSRVLKTAHIDIHK B-CAAF1  DADKDGAVSFEEFVVLVSRVLKTAHIDIHK

Example 2 EN-RAGE (Extracellular Novel-RAGE Binding Protein) ActivatedEndothelial Cells to Mediate Inflammatory Responses

The expression of Receptor for AGE (RAGE) is enhanced in inflammatorysettings such as atherosclerosis and autoimmune vasculitities. Wehypothesized that Receptor for AGE (RAGE) might interact withalternative ligands beyond Advanced Glycation Endproducts (AGEs) in suchsettings. We isolated and purified an ≈12 kDa polypeptide from extractof bovine lung which bore homology to the calgranulin family ofproinflammatory mediators. This polypeptide, called EN-RAGE, bindsimmobilized RAGE and endothelial (EC)/macrophage (MP) RAGE in culturewells with Kd≈75 nM, processes blocked in the presence of anti-RAGE IgGor soluble (sRAGE; the extracellular two-thirds of RAGE). In vitro,exposure of cultured ECs to EN-RAGE increased activation of NF-kB,expression of cell-surface VCAM-1 (4.3-fold compared to treatment withbovine serum albumin BSA), and adhesion of Molt-4 cells (which bearVLA-4, the counter-ligand for VCAM-1)(7-fold compared with BSA), all ina manner inhibited in the presence of anti-RAGE IgG or sRAGE. Exposureof macrophages to EN-RAGE resulted in increased chemotaxis in aRAGE-dependent manner. To test these concepts in vivo, we utilized amodel of delayed hypersensitivity in mice in which footpad injections ofmethylated BSA (mBSA) induce localized inflammation. Pre-treatment(intraperitoneal; IP) with sRAGE prevented mBSA-mediated inflammation ina dose-dependent manner. At 100 μg IP sRAGE, the mBSA-treated footmanifested no inflammation and markedly diminished activation of NF-kBcompared with mice treated with vehicle, mouse serum albumin (MSA);further, elaboration of TNF-alpha into the serum was completelyprevented. Partial anti-inflammatory responses were observed upontreatment of the mice with either anti-RAGE or anti-EN-RAGE F(ab′)2.Nonimmune F(ab′)2 was without effect. Taken together, these findingsindicate that ligands alternative to AGEs such as EN-RAGE activate ECsand MPs, thereby linking RAGE to the generalized inflammatory response.

Example 3 sRAGE Results in Diminished Mortality After Endotoxemia: APotential Treatment for Septic Shock

The use of sRAGE or compounds which are capable of inhibiting theinteraction of EN-RAGE and RAGE could be useful agents for the treatmentof septic shock or sepsis in subjects. It has been shown that a subjectgiven lethal doses of LPS has reduced mortality when the LPS is given inthe presence of sRAGE.

sRAGE and Endotoxemia

Soluble Receptor for AGE (sRAGE) has been shown to prevent inflammationin a model of delayed-type hypersensitivity. Unlike certainanti-inflammatory-type agents, it was believed that sRAGE might exertbeneficial effects when administered in the setting of endotoxemia, aprototypic result of, for example, profound gram negative bacteremia.

When uniformly lethal doses of LPS were administered to Balb/C mice(≈750 μg), administration of sRAGE (pre or post LPS injection) preventeddeath in ≈50% of the mice in pilot studies.

These data underscore the proposition that the potent anti-inflammatoryeffects of sRAGE are not associated with an untoward inclination towardmorbidity/mortality due to the presence of septicemia/endotoxemia.SRAGE, therefore, may be a selective anti-inflammatory agent withselective protective effects against maladaptive inflammatory responses.

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6. Schmidt, A-M, Yan, S-D, Brett, J, Mora, R, Nowygrod, R, and Stern D.Regulation of mononuclear phagocyte migration by cell surface bindingproteins for advanced glycosylation endproducts. J. Clin. Invest.92:2155–2168, 1993.

-   7. Wautier, J L, Chappey, O, Wautier, M P, Hori, O, Stern, D, and    Schmidt A M. Receptor-mediated endothelial dysfunction in diabetic    vasculopathy: sRAGE blocks hyperpermeability. J. Clin. Invest.    97:238–243, 1996.-   8. Miyata, T., Hori, 0, Zhang, J H, Yan, S D, Ferran, L, Iida, Y,    and Schmidt, A M. The Receptor for Advanced Glycation Endproducts    (RAGE) mediates the interaction of AGE-b²-Microglobulin with human    mononuclear phagocytes via an oxidant-sensitive pathway:    implications for the pathogenesis of dialysis-related    amyloidosis. J. Clin. Invest. 98:1088–1094, 1996.-   9. Schmidt, A-M, Hasu, M, Popov, D, Zhang, J-H, Chen, J, Yan, S-D,    Brett, J, Cao, R, Kuwabara, K, Gabriela, C, Simionescu, N,    Simionescu, M, and Stern D. Receptor for advanced glycation    endproducts (AGEs) has a central role in vessel wall interactions    and gene activation in response to circulating AGE proteins.    PNAS(USA) 91:8807–8811, 1994.-   10. Schmidt, A M, Hori, O, Chen, J, Brett, J, and Stern, D. AGE    interaction with their endothelial receptor induce expression of    VCAM-1: a potential mechanism for the accelerated vasculopathy of    diabetes. J. Clin. Invest. 96:1395–1403, 1995.-   11. Lander, H. L., Tauras, J. M., Ogiste, J. S., Moss, R. A.,    and A. M. Schmidt. Activation of the Receptor for Advanced Glycation    Endproducts triggers a MAP Kinase pathway regulated by oxidant    stress. J. Biol. Chem. 272:17810–17814, 1997.-   12. Park, L., Raman, K. G., Lee, K. J., Yan, L., Ferran, L. J.,    Chow, W. S., Stern, D., and Schmidt, A. M. Suppression of    accelerated diabetic atherosclerosis by soluble Receptor for AGE    (sRAGE). Nature Medicine 4:1025–1031, 1998.-   13. Wautier J L, Chappey 0, Wautier M P, Boval B, Stern D and A M    Schmidt. Interaction of diabetic erythrocytes bearing advanced    glycation endproducts with the endothelial receptor RAGE induces    generation of reactive oxygen intermediates and cellular    dysfunction. Circ. 94 (8):#4139, 1996.-   14. Hori, O., J. Brett, T. Slattery, R. Cao, J. Zhang, J. Chen, M.    Nagashima, D. Nitecki, J. Morser, D. Stern, A. M. Schmidt. The    Receptor for Advanced Glycation Endproducts (RAGE) is a cellular    binding site for amphoterin: mediation of neurite outgrowth and    co-expression of RAGE and amphoterin in the developing nervous    system. J. Biol. Chem. 270:25752–25761, 1995.-   15. Yan, S D, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T.    Slattery, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, G.    Godman, D. Stern, and A. M. Schmidt. RAGE and amyloid-b peptide    neurotoxicity in Alzheimer's disease. Nature 382:685–691, 1996.-   16. Yan, S-D., Zhu, H., Fu, J., Yan, S—F., Roher, A., Tourtellotte,    W., Rajavashisth, T., Chen, X., Stern, D. and Schmidt, A-M.    Amyloid-beta peptide-RAGE interaction elicits neuronal expression of    M-CSF: a proinflammatory pathway in Alzheimer's disease. Proc. Natl.    Acad. Sci. 94:5296–5301, 1997.-   17. Slattery, T. K. and Harkins, R. N. Techniques in protein    chemistry IV, ed. Angeletti, R. H., Academic Press, San Diego,    Calif., 1992.-   18. Jaffe, E., Nachman, R., Becker, C., and Minick, R. Culture of    human endothelial cells derived from umbilical veins. Identification    by morphologic and immunologic criteria. J. Clin. Invest.    52:2745–2756, 1973.-   19. Dunn, C. J., Galinet, L. A., Wu, H., Nugent, R. A.,    Schlachter, S. T., Staite, N. D., Aspar, D. G., Elliott, G. A.,    Essani, N. A., Rohloff, N. A., and Smith, R. J. Demonstration of    novel anti-arthritic and anti-inflammatory effects of    diphosphonates. J. Pharmacology and Experimental Therapeutics 266:    1691–1698, 1993.-   20. Wicki, R., Marenholz, I., Mischke, D., Schafer, B. W., and    Heizmann, C. W. Characterization of the human S100A12 (calgranulin    C, p6, CAAF1, CGRP) gene, a new member of the S100 gene cluster on    chromosome 1q21. Cell Calcium 20:459–464, 1996.-   21. Dell'Angelica, E. C., Schleicher, C. H., and Santome, J. A.    Primary structure and binding properties of calgranulin C, a novel    S100-like calcium-binding protein from pig granulocytes. J. Biol.    Chem. 269:28929–28936, 1994.-   22. Wu J, Rogers L, Stern D, Schmidt A M and Chiu D T W. The soluble    receptor for Advanced Glycation Endproducts (sRAGE) ameliorates    impaired wound healing in diabetic mice. Plastic Surgery Research    Council, Abstract #77, p. 43, 1997.

1. A method for inhibiting inflammation in a subject which comprisesadministering to the subject a compound selected from the groupconsisting of (i) an anti-EN-RAGE antibody, (ii) an EN-RAGE-bindingfragment of an anti-EN-RAGE antibody, (iii) the V-domain of soluble RAGEpolypeptide or (iv) an EN-RAGE-binding fragment of the V-domain ofsoluble RAGE polypeptide, thereby inhibiting inflammation in thesubject.
 2. The method of claim 1, wherein the inflammation isassociated with delayed hypersensitivity, accelerated atherosclerosis,or lupus nephritis.
 3. The method of claim 1, wherein the subject is ahuman, a primate, a mouse, a rat or a dog.
 4. The method of claim 1,wherein the administration comprises intralesional, intraperitoneal,intramuscular or intravenous injection; infusion; liposome-mediateddelivery; or topical, intrathecal, per rectum, gingival pocket,intrabronchial, nasal, oral, ocular or otic delivery.
 5. The method ofclaim 1, wherein the compound is administered hourly, daily, weekly,monthly or annually.
 6. The method of claim 1, wherein the effectiveamount of the compound comprises from about 0.000001 mg/kg body weightto about 100 mg/kg body weight.
 7. The method of claim 1, wherein thesubject is suffering from systemic lupus erythematosus, inflammatorylupus nephritis, septic shock or endotoxemia.
 8. The method of claim 1,further comprising administering to the subject a pharmaceuticallyacceptable carrier during the administration of the compound.
 9. Themethod of claim 8, wherein the carrier comprises a diluent.
 10. Themethod of claim 8, wherein the carrier comprises a virus, a liposome, amicroencapsule, a polymer encapsulated cell or a retroviral vector. 11.The method of claim 8, wherein the carrier is an aerosol, intravenous,oral or topical carrier.
 12. The method of claim 8, wherein the compoundis administered from a time release implant.
 13. The method of claim 1,wherein the subject is suffering from an autoimmune or inflammatorydisorder in which the recruitment of EN-RAGE-containing inflammatorycells occurs.
 14. The method of claim 1, wherein the subject issuffering from a bacterial-associated or other pathogen-associatedinfection.
 15. The method of claim 1, wherein the antibody is amonoclonal antibody or a polyclonal antibody.
 16. The method of claim 1,wherein the antibody is a chimeric antibody, a humanized antibody or aprimatized antibody.
 17. The method of claim 1, wherein the compound isan anti-EN-RAGE antibody.
 18. The method of claim 1, wherein thecompound is an EN-RAGE-binding fragment of an anti-EN-RAGE antibody. 19.The method of claim 1, wherein the compound is the V-domain of solubleRAGE polypeptide.
 20. The method of claim 1, wherein the compound is anEN-RAGE-binding fragment of the V-domain of soluble RAGE polypeptide.